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This prospective multicenter sentinel lymph node (SLN) trial investigated whether molecular analysis would improve the detection of SLN metastases and their prognostic value. We report mammaglobin quantitative real-time polymerase chain reaction (qRT-PCR) results and clinical outcome for 547 patients (mean follow-up 7 years). Breast cancer patients (excluding stage IV disease or palpable nodes) were enrolled from 1996 to 2005 at 16 institutional review board-approved sites. Alternate 2-mm serial sections of each SLN were examined by hematoxylin and eosin staining with or without immunohistochemistry at multiple levels or blinded and assayed by Taqman qRT-PCR according to previously established thresholds. Mammaglobin remains a highly specific (99%), sensitive (97% primary tumor; 82% N1 SLN) marker for breast cancer. Mammaglobin SLN expression was associated with other prognostic factors, was detected in most patients with distant recurrence (48 of 79; 61%), and was associated with decreased recurrence-free survival (log rank P

Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8(+) cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8(+) CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8(+) CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8(+) CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8(+) CTL lines lysed the HLA-B7(+)/MGBA(+) human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7(-) or MGBA(-) breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8(+) CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.

Breast cancer (BC) is the most frequent malignant tumor in women worldwide; its recurrence is a result of undetected metastasis present at the time of primary patient treatment. The detection of cell-free RNA in plasma and serum of human subjects has found increasing applications in the field of medical diagnostics.This study aimed at evaluating plasma mammaglobin mRNA as a useful tumor marker for the diagnosis and the detection of metastasis in breast cancer at the time of diagnosis either alone or in combination with conventional serum tumor markers CA15.3 and/or CEA. This study included 40 Egyptian females with primary breast cancer and 25 with different benign breast diseases. The BC group was classified into 24 patients with localized BC and 16 patients with metastases. All patients were subjected to routine laboratory investigations, estimation of serum CA15.3 and CEA by electrochemiluminescence immunoassay and detection of plasma mammaglobin mRNA by using nested reverse transcriptase polymerase chain reaction (RT-PCR). There was a significance increase in plasma mammaglobin mRNA expression, CA15.3 and CEA levels in BC group as compared to benign group (p0.001). No significant difference was observed between levels of plasma mammaglobin mRNA expression in patients with tumor's size or grade. No correlation was observed with plasma mammaglobulin mRNA levels and tumor size or grading, CEA and CA15.3. There was a significant difference (p0.05) and a positive correlation between CA 15.3 and CEA levels in patients with tumor size and grading. Expression of plasma mammaglobin mRNA has the highest sensitivity, specificity and diagnostic accuracy for both BC and BC with metastasis (75%, 92%81.5%) and (87.5%, 45.8%62.5%), respectively. To improve diagnostic efficacy of BC, the use of combined tests, expression of plasma mammaglobin mRNA and CA 15.3 improved the sensitivity, specificity and diagnostic accuracy to 90%, 80%86.2%, respectively; as well as in BC with metastasis to 100%, 79.2%,87.5%, respectively. In conclusion, plasma mammaglobin mRNA alone or in combination with CA15.3 may be used as a valuable noninvasive approach for the diagnosis and the detection of metastasis in breast cancer at the time of diagnosis.

Detection of residual tumor cells in the circulation can provide prognostic as well as therapeutic information and help in identifying patients at high risk for developing metastases. Maspin and mammaglobin are two molecules that are specifically associated with breast cancer. We looked for mammaglobin and maspin transcripts in the peripheral blood of patients with breast cancer and evaluated their utility as a marker of the response to therapy. Maspin and mammaglobin transcripts were analyzed in 85 breast-cancer patients by nested RT-PCR, prior to and after treatment. Before therapy, 10 patients were found positive for mammaglobin and 20 patients were positive for maspin. In four patients, both transcripts were detected. Immediately following treatment, only one patient was still positive for mammaglobin while maspin transcripts persisted in three patients. Disease progression was observed mainly in patients in whom maspin transcripts were not detectable. Molecular detection of circulating tumor cells during therapy based on analysis for mammaglobin and maspin transcripts is an easy and practical method that can be applied to follow-up patients. We suggest that detection of mammaglobin mRNA is useful to determine the effect of therapy while maspin transcripts may indicate more aggressive disease.

Sekretoglobuline wie Mammaglobin 1 (MGB1) und Lipophilin B (LipB) sind kleine, selten glykosylierte Proteine, deren Expressionsmuster vorwiegend im sekretorischen Epithel von Säugetieren nachweisbar sind. Diese liegen als Homo- bzw. Heterodimere vor, deren genaue physiologische Bedeutung bis dato unbekannt ist. Allerdings scheinen einigen Vertretern der SCGBs eine funktionelle Bedeutung in der Genese diverser Tumore zuzukommen. Die Expression von LipB sowie MGB1 ist im humanen Mammakarzinom hochreguliert. Eine deutliche Hochregulation konnte in einer transgenen Brustkrebszelle (7G4) gezeigt werden, die die mGPx4, ein reduzierendes Enzym mit anti-apoptotischen Eigenschaften, überexprimiert. MGB1 ist ein relativ gut erforschter Tumormarker für Brustkrebs. Dagegen ist nur sehr wenig über die Expressionsregulation seines Dimerpartners LipB bekannt. Zu klärende Kernpunkte dieser Arbeit waren deshalb zum einen die Beleuchtung der transkriptionellen Aktivierung der LipB-Expression. Zum anderen die Diskussion der möglichen biologischen und mechanistischen Funktion der mGPx4, die mit einem veränderten Expressionsmuster im Brustkrebs assoziiert ist. Zur Klärung der erst genannten Problematik konnten initiale in silico-Analysen cis- aktivierende Elemente im proximalen LipB-Promotor identifizieren. Funktionelle Promotorstudien bestätigten innerhalb dieser Sequenz deutliche transkriptionelle Aktivitäten. Daran anschließende EMSA-Versuche zeigten spezifische DNA-Protein-Interaktion der Faktoren Sp1 sowie NF1. Jedoch konnten darin keine differentiellen Gelshifts nachgewiesen werden. Die Untersuchungen der potentiellen Beteiligung der mGPx4 in die Transkriptelongation und –stabilität ergaben keine signifikanten Unterschiede zwischen 7G4 und VC. Die Erforschung epigenetischer Mechanismen wie der CpGMethylierung und Histonacetylierung waren in ihrer Aussage uneinheitlich. 5-Aza-2’- desoxycytidin, ein Methyltransferase-Inhibitor, bewirkte eine generelle transkriptionelle Aktivierung. TSA und Butyrat dagegen reprimierte die MGB1/LipBExpression. Schließlich gaben real time-PCR-Analysen Hinweise auf den Zusammenhang von Proliferationsarrest und HDACI-induzierte p21WAF1/CIP1-Hochregulation., Mammaglobin A (MGB1) and lipophilin B are (LipB) members of the secretoglobin superfamily, a group of small, secretory, rarely glycosylated, dimeric proteins with unclear physiologic functions, mainly expressed in mammalian secretory epithelia. It seems that the secretoglobin polypeptides could be of functional importance in the development of various human cancers. Moreover, the expression levels of mammaglobin A in breast tumors were up-regulated and significantly correlated with those of lipophilin B. An explicit up-regulation was shown in a gene-modified mamma carcinoma cell (7G4), which overexpressed the mitochondrial GPx4, a reductive and anti-apoptotic protein. Since Mammaglobin A is a well-known marker for breast tumor, there are some studies about his regulation and expression, but on the other hand it is still little known about the gene regulation of its dimeric partner Lipophilin B. The aims of the present study are on the one hand to investigate the transcriptional activation of the LipB gene expression in 7G4 and control cells (vector control). On the other hand the discussion about the possible biological and mechanistic relation of GPx4 associated with the changed expression pattern of secretoglobins in breast cancer. Initial in silico-analysis of the proximal LipB-up streamsequenz could identify cis-acting elements. Luciferase reporter assays confirm transcriptional activities in this promoter region. Following studies of end-labeled DIG probes in EMSAs demonstrated specific DNA-protein- interactions with the maximum likelihood, indicating the binding of Sp1 and NF1. However no differential shift binding was observed. The analysis of the involvement of the mitochondrial GPx4 in transcript stability and elongation revealed no significant differences. Elucidating epigenetic regulatory mechanisms (e.g. DNA Methylation and Histone Acetylation) addicted inconsistent conclusions. The use of 5-Aza-2’-desoxycytidine confirmed general transcriptional activity, whereas Trichostatin A and Butyrat could downregulate MGB1/LipB gene expression in mGPx4 transfectant and vector control. In addition, real time PCR analysis revealed the relation of cell growth arrest and the up regulation of p21WAF1/CIP1 due to TSA/Butyrat application.

Mammaglobin A is a specific marker of the normal and neoplastic mammary tissue that usually is detected by RT-PCR. Few data are available about the immunohistochemical expression of this marker in mammary carcinoma and about the significance of the positive reaction. Our purpose was to investigate the sensitivity of the mammaglobin expression in breast cancer and to determine its correlations with conventional prognostic parameters. There were investigated 47 patients with breast carcinoma, and slides from paraffin blocks were stained with an antibody against mammaglobin. The immunohistochemical reaction was scored based on the percentage of positive tumor cells in both primary tumors and lymph node metastasis. Positive reaction for mammaglobin was found in the normal mammary tissue adjacent to the tumor in all cases, in 78.72% primary breast carcinoma, and in 58.06% of cases with lymph node metastases. A significant correlation was found between the mammaglobin expression in the primary tumor, grade, and lymph node status, but not with the age of the patient, pathologic subtype of carcinoma and stage of the tumor. The ductal in situ carcinoma associated to the invasive tumor did not influence significantly the prognostic value of mammaglobin expression. Out results suggest that mammaglobin is a sensitive marker of breast carcinoma, it defines a subgroup of patients with better prognosis and is a useful method to detect breast cancer metastases.

Sentinel lymph node biopsy (SLNB) is becoming accepted as standard of care for axillary staging in breast cancer. World Health Organization (WHO) re-classification of axillary metastases into macrometastases, micrometastases and individual tumour cells has highlighted the issues of sampling and further histological examination of the initially negative SLNB. Molecular detection of metastatic breast cancer cells in lymph nodes is now available as a commercial kit for intraoperative use and can resolve the sampling issue. Semiquantitative assessment of axillary lymph node tumour burden can now be made using two separate technologies (histology and reverse transcriptase-polymerase chain reaction). The clinical implications of low metastatic axillary lymph node tumour burden are not clear, and future trials need to include molecular data. The consequences of the availability of molecular assessment are reviewed.

Background: Bone marrow (BM) biopsy has been suggested as an independent prognostic factor in patients with breast cancer. Methods: Patients operated for breast cancer from June 2000 to April 2008 were enrolled in this protocol after signing an informed consent. After primary surgery, BM aspirate from the iliac crest was obtained and 5-10 cc of blood collected. Since 2002 a peripheral blood (PB) sample was also obtained. Both carcinoembryonic antigen (CEA) and Mammaglobin-specific nested reverse-transcription polymerase chain reaction (RT-PCR) were used to examine BM and PB samples. Physicians and patients were blinded to results. Results: Two hundred seventy-three patients underwent BM and/or PB test. The median age of the patients was 63 years (31-80 years), and the median tumor diameter was 1.5 cm (0.1-6 cm). BM aspirates were unsuccessful in nine patients, and RT-PCR was not technically feasible in 18 women, leaving 246 patients available for analysis of results and follow-up. Among them, 110 patients (45%) had either a BM or a PB test positive for CEA or Mammaglobin (Test+). At median follow-up of 60 months, 31 events (deaths or relapse) occurred (13%). Disease-free survival (DFS) was significantly lower in the Test+ group (BP and/or PB) (P < 0.001). This effect was independent of nodal status. At 5 years, event-free survival for Node-/Test- patients was 46/49 (94%) and for Node+/Test+ patients was 21/33 (64%), while patients with only one status positive (Node-/Test+ or Node+/Test-) had an intermediate disease-free survival (35/43, 81%) (P = 0.005). In a subgroup analysis, RT-PCR results for BM and Mammaglobin retained statistical significance on DFS (P < 0.001), while those for PB and CEA did not. Conclusions: This study confirms that RT-PCR of the BM is an independent prognostic factor for disease-free survival of breast cancer patients, and may improve their staging, allowing better strategies for therapy and follow-up. © 2009 Society of Surgical Oncology.

Morphologic differentiation of breast carcinoma from nonmammary malignancies in fluid specimens can be a diagnostic challenge. Immunocytochemistry is often employed in the differential diagnosis. In this study, we evaluated the expression of mammoglobin (MGB1) in body-cavity fluid specimens and compared its efficacy as a marker for metastatic breast carcinomas with that of gross cystic disease fluid protein-15 (GCDFP-15). Cell blocks from 40 fluid specimens were immunostained with monoclonal antibodies against MGB1 and GCDFP-15. They included 15 breast carcinomas and 25 nonmammary carcinomas (10 lungs, 10 ovaries, 3 gastrointestinal tracts, 1 kidney, and 1 urinary bladder). Positivity was defined as the presence of cytoplasmic staining in 10% or more carcinoma cells. Thirteen (87%) and seven (47%) breast carcinomas showed positive staining with MGB1 and GCDFP-15, respectively. Three (12%) nonmammary carcinomas (2 ovarian and 1 colonic) showed positive MGB1 staining; one (3%) nonmammary carcinoma demonstrated positive GCDFP-15 staining. The differences of MGB1 and GCDFP-15 staining between breast and nonmammary carcinomas were statistically significant (P < 0.05). Both MGB1 and GCDFP-15 are specific markers for metastatic breast carcinomas in cell block fluid specimens (88 vs. 96%). However, MGB1 is more sensitive than GCDFP-15 as a marker for metastatic breast carcinoma (87 vs. 46%).

The distinction of primary lung from metastatic breast cancer is crucial in patients presenting with a solitary pulmonary nodule after mastectomy, because treatment strategies are completely different. Definitive diagnosis of these nodules, however, is often difficult. We assessed the feasibility of our diagnostic approach for these nodules and estimated the frequency of primary lung cancer occurrence in patients after mastectomy.We evaluated solitary pulmonary nodules appearing in 48 patients after mastectomy. For histological examination, CT-guided needle aspiration biopsy (CT-NAB) or trans-bronchial lung biopsy (TBLB) was performed. Besides conventional morphopathological examination, differential diagnosis was performed by immunohistochemical examination and evaluation using a molecular marker (mammaglobin 1).Biopsy specimens were obtained using minimally invasive methods, namely CT-NAB and TBLB, in 91.7% of patients. From 48 patients, differential diagnosis was obtained by morphopathological methods alone in 32, and by immunohistochemical and molecular marker examination in the remaining 16. Final diagnosis was metastatic breast and primary lung cancer in 40 (83.3%) and 8 patients (16.7%), respectively.Our results show the clinical feasibility of our stepwise approach to the differential diagnosis of primary lung cancer and breast cancer relapse presenting as a solitary nodule in patients after mastectomy.

Differentiation of Müllerian serous carcinoma from metastatic breast carcinoma is a challenging and frequent diagnostic dilemma, particularly in the setting of a pelvic mass or peritoneal carcinomatosis. Precise classification is important as it impacts treatment and prognosis. Antibodies exist that assist with this differential but they are often limited by low sensitivity or specificity. This study evaluated the utility of mesothelin and mammaglobin antibodies in differentiating breast carcinoma (particularly those with a papillary morphology) from Müllerian serous carcinomas. Formalin-fixed, paraffin-embedded archival tissue from 21 breast carcinomas (10 micropapillary, 11 usual type ductal carcinomas) and 20 serous carcinomas (12 ovarian and 8 uterine) in addition to 6 cases of metastatic breast cancer to the ovary (5 cases) and cervix (1 case) were evaluated for the pattern and intensity of reactivity to antibodies to mesothelin, mammaglobin, and GCDFP-15. None of the breast carcinomas stained for mesothelin, whereas 8/12 and 3/8 ovarian and uterine serous carcinomas were positive; however, 7 of these had less than 10% positivity. Mammaglobin was negative in all serous carcinomas. When compared with GCDFP-15, mammaglobin was more frequently and diffusely expressed in breast carcinomas (GCDFP-15 positivity in 8/21 and mammaglobin positivity in 14/21). This study indicates that the addition of mammaglobin to immunohistochemical panels is useful in distinguishing metastatic breast carcinoma from a new primary ovarian or uterine malignancy. Mesothelin is extremely specific in this scenario but can be technically challenging to interpret due to the common patchy, focal staining.

Background. Intra-operative assessment is not routinely performed in the UK due to poor sensitivity of available methods and overburdened pathology resources. We conducted a prospective clinical feasibility study of the GeneSearch™ Breast Lymph Node (BLN) Assay (Veridex, LLC, Warren, NJ) to confirm its potential usefulness within the UK healthcare system. Methods In the assay 50% of the lymph node was processed to detect the presence of cytokeratin-19 and mammaglobin mRNA. The assay was calibrated to detect metastases >0.2 mm. Assay results were compared to H&E performed on each face of ~2 mm alternating node slabs and 3 additional sections cut at ~150 μm interval from each face of the node slab. Results 124 sentinel lymph nodes were removed from 82 breast cancer patients. The assay correctly identified all 6 patients with sentinel node macrometastases (>2.0 mm), and 2 of 3 patients with sentinel node micrometastases (0.2–2.0 mm). Sentinel lymph nodes in 4 patients were assay positive but histology negative. Two of these four patients had isolated tumor cells seen by histology. The overall concordance with histology was 93.9% (77/82), with sensitivity of 88.9% (8/9, 95% CI 56.5–98%), specificity of 94.6% (69/73, 95% CI 86.7–97.8%), positive predictive value of 66.7% (8/12, 95% CI 39.1–86.2%) and negative predictive value of 98.6% (69/70, 95% CI 92.3–99.7%). The assay was performed in a median time of 32 min (range 26–69 min). Conclusion Intra-operative assessment of sentinel lymph node can be performed rapidly and accurately using the GeneSearch™ BLN Assay.

Mammaglobin (SCGB2A2) and lipophilin B (SCGB1D2) are members of the secretoglobin polypeptide family. Mammaglobin has been shown to be overexpressed in breast tumor tissue, indicating that mammaglobin might confer a growth advantage to mammaglobin-expressing tumor cells.The mammaglobin and lipophilin B mRNA expression levels were investigated in seven breast tumors and matched nonneoplastic tissues from the same patients using quantitative real-time RT-PCR. The effect of mammaglobin and lipophilin B expression on breast cancer cell proliferation rates was investigated by analyzing retrovirally transduced Hs578T cell clones. Cell proliferation rates were determined during the exponential growth phase by analyzing the change in lactate dehydrogenase activity over time.All analyzed breast cancer tumors had lower expression levels of mammaglobin and lipophilin B than the respective mean level of the nonneoplastic breast tissues; no prominent overexpression was evident. There was high variability in the expression of mammaglobin and lipophilin B among the non-neoplastic samples, showing that caution should be taken when evaluating their over- and underexpression in tumors. The expression levels of mammaglobin and lipophilin B correlated with each other in the analyzed samples (p = 0.001). Ectopic overexpression of mammaglobin and lipophilin B did not affect the cell proliferation rate of Hs578T breast carcinoma cells in vitro.Our findings suggest that the overexpression of mammaglobin observed in certain breast tumors is an epiphenomenon not causally involved in breast carcinogenesis.

Mammaglobin (MGB), a secretory protein belonging to the uteroglobin/Clara cell protein family, is a sensitive marker for breast carcinoma, but is also reported to be expressed in the female genital tract and its neoplasms. Details of MGB expression pattern and its pathologic significance in the female genital tract have not been systematically studied. To investigate the potential use of MGB in gynecologic pathology practice, we tested MGB expression by immunohistochemistry on 47 endocervical adenocarcinomas (whole tissue sections of 13 invasive and 35 in situ) and 55 endometrial carcinomas (39 endometrioid and 16 nonendometrioid represented on a single tissue microarray). Nonneoplastic endocervical and endometrial tissues were also evaluated for MGB expression. MGB expression was detected in thirty (77%) of 39 of endometrioid endometrial adenocarcinomas compared with 4 (31%) of 13 endocervical adenocarcinomas. MGB was mostly negative in nonendometrioid endometrial carcinoma (negative in 14 [88%] of 16). Endocervical adenocarcinoma in situ (AIS) showed either weak (predominantly) or moderate (occasionally) expression in about 40% of the cases in comparison with strong positivity in benign endocervical glandular epithelium. Reduction of MGB staining was seen in transition from benign epithelium to AIS. These results confirm that MGB is not specific for breast carcinoma, but is also variably expressed in nonneoplastic and neoplastic endocervical and endometrial tissues. Frequent MGB expression in endometrioid endometrial adenocarcinoma is significantly different from nonendometrioid carcinoma. Hormone receptor status is not associated with MGB expression in endometrial carcinomas. Most endocervical adenocarcinomas are negative for MGB, in contrast to mostly positive endometrioid endometrial adenocarcinomas, however, MGB expression alone is not specific enough to distinguish these 2 tumor types. MGB expression is altered in neoplastic endocervical epithelium compared with normal, and may indicate its decreased expression in the process of early carcinogenesis. MGB may be a promising new adjunctive marker in gynecologic pathology.

Leptomeningeal (LM) carcinomatosis is an increasing clinical complication in patients with advanced breast cancer (BC). The LM carcinomatosis diagnostic procedures rely mainly on cerebrospinal fluid (CSF) cytology, although both the amount of CSF and the number of malignant cells remain limiting factors. Therefore, efforts should be made to design new highly sensitive diagnostic tools to detect malignant cells in CSF of BC patients with LM carcinomatosis. In this study, the human Mammaglobin (hMAM) mRNA amplification by RT-PCR was employed to detect metastatic cells in CSF and thus, to diagnose LM carcinomatosis in a BC patient. Our data demonstrate that hMAM transcripts are expressed in the CSF of a BC patient with LM carcinomatosis, hence making RT-PCR for hMAM a potentially suitable test to identify occult BC cells in the brain.

Background The accuracy of a molecular reverse transcriptase–polymerase chain reaction (RT-PCR)–based assay for metastases detection in axillary sentinel lymph nodes (SLNs) has recently been validated in our institution and adopted as an intraoperative test for breast cancer patient management. Methods Molecular assay performance was compared to standard postoperative histology in 253 consecutive patients with clinically node-negative T1 early breast cancer ( Results The molecular assay correctly identified 26/27 macrometastases and 11/15 micrometastases. Overall concordance with histopathology was 93%, with 87% sensitivity, 94% specificity, and 75% positive and 97% negative predictive values. The molecular assay was positive in 13/14 patients with SLNs and nonsentinel lymph node (axillary lymph node [ALN])-positive histology. Notably, 2/12 patients with assay-positive/histology-negative SLNs exhibited ALN positivity. Conclusions This molecular assay can raise the standard of care for patient management as its accuracy is similar to that of standard postoperative histology with the advantage of being standardized, objective, and fast enough for intraoperative use.

Objectives The prognosis of breast cancer patients depends on primary tumor resection and axillary lymph nodes examination. The purpose of this study was to analyze by molecular biology techniques the presence of mammaglobin A and B messenger RNA in breast sentinel lymph node (SLN) by reverse-transcription polymerase chain reaction (RT-PCR). Methods Sentinel lymph nodes from 50 patients with a diagnosis of breast cancer were prospectively studied between June 2004 and August 2006. Lymph nodes were all examined every 2 mm by intraoperative cytology. Hematoxylin-eosin (HE), immunohistochemistry (IHC) with cytokeratin (clone AE1-AE3, DAKO, dilution 1:100), and molecular biology techniques were used in all cases. Results Deferred study with routine techniques showed subcapsular metastasis in 3/50 cases. Out of 50 cases, 5 were detected with IHC, and 2 of them were negative for HE. Multiplex RT-PCR allowed the detection of 18/50 positive SLN, which included the 5 above-mentioned cases. The other SLN studied (32/50) showed no metastases with the methods herein implemented. Conclusions The epidemiologic impact of incomplete SLN study has been observed, as the HE technique fails to identify all SLN with micrometastases. In our opinion, SLN should be studied with IHC and molecular biology techniques. The multiplex RT-PCR technique for A and B mammaglobin proves to be specific and sensitive. This study will serve to formulate hypotheses. Further research, including a larger population and a longer-term follow-up period, will be required to confirm these hypotheses. Should our findings be confirmed in the future, molecular biology determinations could modify patients' staging and treatment.

Background In breast cancer patients, the status of the sentinel lymph nodes (SLNs) has been shown to accurately reflect the presence of metastases in the axillary lymph nodes (ALNs). Intra-operative SLN evaluation by frozen section histology may miss positive cases, leading to a second surgery for complete ALN dissection. Permanent section histology itself has tissue sampling limitations and is partially dependent on pathologist expertise. Methods A prospective study ( N =78) was conducted in our institution to validate a new intra-operative molecular assay, the GeneSearch™ breast lymph node (BLN) assay. This assay quantifies the expression of mammaglobin and cytokeratin-19 genes using quantitative RT-PCR technology to determine SLN status. Fresh SLN sections (2mm thick) were analyzed alternatively by BLN assay or post-operative histology (haematoxylin–eosin and immunohistochemistry). The subject was considered positive when histology revealed a focus >0.2mm. Results BLN assay results corroborated with histologic results in 75 out of 78 patients for an overall agreement of 96%, a sensitivity of 92%, and a specificity of 97%. The positive and negative predictive values of the BLN assay were of 86% (12/14) and 98% (63/64), respectively. Interestingly, a statistically significant correlation was observed between the metastases' histologic size and both assay markers' expression levels as represented by cycle time to positivity ( ρ ≥0.71, all p Conclusions The performance of the BLN assay in identifying nodal metastases >0.2mm was similar to that of permanent section histology, with the added advantages of an objective and rapid output that could be used for intra-operative decision to remove additional ALN.

To assess the accuracy of a commercially available real-time reverse-transcription-polymerase chain reaction assay for mammaglobin and cytokeratin 19 mRNAs [GeneSearch Breast Lymph Node (BLN) Assay, Veridex LLC, Warren, NJ] in the detection of axillary sentinel lymph nodes (SLNs) metastases in patients with breast carcinoma.Because of the lack of standardized and widely accepted protocols for a truly accurate histopathologic examination of SLN, the relative merits of alternative assays based on the identification of tumor specific mRNA markers deserve further assessment.: A prospective series of 293 consecutive SLNs from 293 patients was evaluated. The BLN assay results were compared with those of an extensive histopathologic examination of the entire SLNs performed on serial frozen sections cut at 40 to 50 microm intervals.The BLN assay correctly identified 51 of 52 macrometastatic and 5 of 20 micrometastatic SLNs, with a sensitivity of 98.1% to detect metastases larger than 2 mm, 94.7% for metastases larger than 1 mm, and 77.8% for metastases larger than 0.2 mm. The overall concordance with histopathology was 90.8%, with specificity of 95.0%, positive predictive value of 83.6%, and negative predictive value of 92.9%. When the results were evaluated according to the occurrence of additional metastases to non-SLN in patients with histologically positive SLNs, the assay was positive in 33 (91.7%) of the 36 patients with additional metastases and in 22 (66.6%) of the 33 patients without further echelon involvement.The sensitivity of the reverse-transcription -polymerase chain reaction assay is comparable to that of the histopathologic examination of the entire SLN by serial sectioning at 1.5 to 2 mm.

Mammaglobin-A(mam-A) is expressed in over 80% of human breast tumors. We recently reported that mam-A DNA vaccination resulted in breast cancer immunity in a preclinical model. Here we investigated whether mam-A HLA-class-I tetramers could be used to monitor and define the role of CD8(+)cytotoxic T-lymphocytes(CTL) in mediating breast cancer immunity following mam-A DNA vaccination.Mam-A DNA vaccination was performed in HLA-A2(+)huCD8(+ )transgenic mice. HLA-A2 tetramers carrying the immunodominant mamA2.1 peptide were used to monitor CD8(+)CTL. Human breast cancer colonies were developed in immunodeficient SCID-beige mice. ELISPOT was used to correlate frequency of mamA2.1 tetramer(+)CD8(+)T cells and IFN-gamma production [spots per million cells (spm)] in human subjects.Vaccination of HLA-A2(+)huCD8(+) mice with mam-A DNA vaccine, but not empty vector, led to the expansion of mamA2.1 tetramer(+)CD8(+)T-cells in peripheral blood (0.5% pre-vaccination compared to2.0% post-vaccination). CD8(+)T cells from vaccinated mice specifically lysed UACC-812(HLA-A2(+)/mam-A(+), 25% lysis) but not MDA-MB-415(HLA-A2(-)/mam-A(+)) or MCF-7(HLA-A2(+)/mam-A(-)) breast cancer cells. Adoptive transfer of purified CD8(+)T cells from vaccinated mice into immunodeficient SCID-beige mice with established human breast cancer colonies led to tetramer(+)CD8(+ )T-cell infiltration with regression of UACC-812 but not MCF-7 tumors. HLA-A2(+) breast cancer patients revealed increased frequency of mamA2.1 tetramer(+)CD8(+ )T-cells compared to normal controls (2.86 +/- 0.8% vs. 0.71 +/- 0.1%, P = 0.01) that correlated with the IFN-gamma response to mamA2.1 peptide (48.1 +/- 20.9 vs. 2.9 +/- 0.8 spm, P = 0.03).CD8(+ )T-cells are crucial in mediating breast cancer immunity following mam-A DNA vaccination. Mam-A HLA-class-I tetramers can be effectively used to monitor development of CD8(+ )T-cells following mam-A vaccination.

Gross cystic disease fluid protein (GCDFP-15) and mammaglobin are both widely used and accepted markers for epithelia of breast origin. We aimed to evaluate their relation of expression on parallel whole tissue sections in primary breast cancer by immunohistochemistry and also to correlate it with clinico-pathological parameters including patient survival. Primary breast carcinomas from 165 patients with a mean clinical follow-up of 73 months were immunostained using commercially available antibodies against GCDFP-15 and mammaglobin. An immunoreactive score (IRS) was calculated based on the cytoplasmic staining intensity and the number of cells stained. Cytoplasmic expression of GCDFP-15 and mammaglobin was observed in 73.3% and 72.1% of invasive breast carcinomas respectively. 91.8% of breast cancer cases expressed at least one of both markers. Both markers strongly correlated with each other and were significantly associated with lower tumour grading. Additionally, GCDFP-15 negativity was significantly associated with shortened disease-free survival times in univariate and multivariate analyses. We demonstrated the strong correlation of GCDFP-15 and mammaglobin with each other and showed that only very few primary breast cancers are completely negative for both markers. The significantly longer disease free survival times for patients with GCDFP-15 positive tumours clearly warrants further study.

To investigate the expression of the human mammoglobin (hMAM) mRNA in bone marrow and its clinical significance in the breast cancer patient.Expression of hMAM mRNA was detected using nested reverse transcription polymerase chain reaction (RT-PCR) in the bone marrow aspiration sample from 75 breast cancer patients, 15 patients with benign breast lesions and 8 healthy volunteers as control. The possible correlation of hMAM mRNA expression with clinico-pathological parameters and related molecular markers such as Ki67, p53 and VEGF were analyzed.The sensitivity of RT-PCR in this series reached 10(-6). The hMAM mRNA was found to be positively expressed by RT-PCR in 21 of 75 breast cancer patients with a positive rate of 28.0%. However, hMAM mRNA expression was not detected in the bone marrow aspiration samples from patients with benign breast lesions and healthy volunteers. The hMAM mRNA expression was positively correlated with axillary nodal involvement and progesterone receptor (PR) status (P0.05) as well as Ki67 expression in breast cancer tissue (chi2 = 4.936, P = 0.026), but not with age, tumor size, clinical stage, or estrogen receptor (ER) status (P0.05).RT-PCR is quite sensitive and has a high specificity in detecting the presence of hMAM mRNA in the bone marrow from breast cancer patients. Thereupon, hMAM mRNA may be useful as a molecular biomarker in detecting disseminated tumor cells (DTC) in the bone marrow of breast cancer patients. Positive hMAM mRNA expression result may have an impact upon therapeutic recommendations and patients' prognostic judgement.

Mammaglobin-A (MGB) is a breast cancer-associated antigen that is an attractive target for immune intervention. MGB has been shown to induce a specific CD8 T cell response in breast cancer patients, but little is known about a possible MGB-specific CD4 T cell response.Peripheral blood-derived CD4(+)CD25(-) T cells were stimulated in vitro with MGB-pulsed antigen-presenting cells (APC). The MGB and human leukocyte antigen (HLA) class II specificity of the CD4 T cell lines was confirmed by cytokine release following restimulation with autologous and allogenic APC pulsed with MGB from different sources. Candidate HLA class II-restricted epitopes were identified by computer algorithm and validated in cytokine release assays.MGB-specific CD4 T cells were successfully generated in cultures from six of seven donors. Restimulation of MGB-specific CD4 T cells with MGB-pulsed APC induced significantly higher levels of interferon (IFN)-gamma release than APC pulsed with an irrelevant protein (P = 0.0004). Cultures from five of seven donors showed a pure Th1 type response as evidenced by the absence of interleukin (IL)-4. MGB-specific CD4 T cells recognized both recombinant and naturally processed MGB presented by APC. This recognition was HLA class II-restricted, as HLA-DR mismatched APC were not recognized. MGB-specific CD4 T cells from three of four donors recognized MGB-derived, HLA class II-restricted peptides pulsed onto APC.We have successfully generated MGB-specific CD4 T cell cultures and identified candidate MGB HLA class II epitopes. These studies should facilitate study of the CD4 T cell response to MGB, and the development and monitoring of vaccine strategies targeting this unique antigen.

Previously, we used the reverse transcription-polymerase chain reaction (RT-PCR) to show that mammaglobin (MGB1) can serve as a differential marker of breast cancer metastasis from primary lung cancer. However, mRNA-based methods are not appropriate for use in clinical practices. In this study, we examined MGB1 protein expression in 480 tumors from various organs using immunohistochemical detection and a tissue microarray technique. Breast cancers expressing MGB1 were also analyzed clinicopathologically to determine whether these cancers constitute a characteristic subset. Immunohistochemically, MGB1 was expressed specifically in breast cancers. Of the other cancers examined, including 29 of the head and neck, eight of the thyroid, 106 of the lung, 35 of the gastrointestinal tract, three of the pancreas, 14 of the uterine cervix and 13 of the ovary, none were positive for MGB1 except a proportion of salivary gland tumors (6/11, 55%) and endometrial cancers (3/23, 13%). Among the 238 breast cancers, MGB1 was expressed in 114 (48%), most of which were classified histologically as invasive duct or lobular carcinomas. Clinicopathologically, MGB1 expression was associated with positive expression of estrogen receptors and negative expression of CK5, but not with pathological stage, HER2 gene amplification or p53 immunoreactivity. Kaplan-Meier analysis revealed prolonged disease-free survival in patients with MGB1-positive breast cancers (log rank test, P=0.016), but the Cox proportional hazard model failed to confirm that MGB1 was an independent prognostic factor (hazard ratio 1.77, P=0.1755). In terms of practical diagnosis, MGB1 immunohistochemistry can serve as a differential marker of breast cancer metastasis from primary lung cancer for two reasons. Firstly, HER2-positive breast cancer frequently lacks estrogen receptor expression, but MGB1 is expressed in about half of this subtype. Secondly, as primary lung adenocarcinomas may express estrogen receptors, MGB1 expression provides further discrimination of the origin of breast cancers.

There are limited data that compare the usefulness of mammaglobin with gross cystic disease fluid protein-15 (GCDFP-15) in the identification of breast carcinomas. Whole tissue sections of 29 breast carcinomas with matched lymph node metastases and 63 breast carcinomas on tissue microarray were stained with mammaglobin cocktail and GCDFP-15 antibodies. In addition, tissue microarrays (US Biomax, Rockville, MD) containing 544 different human tumors were also stained with the mammaglobin antibody cocktail. Positive staining was seen in 67 (55.4%) of 121 breast carcinomas with mammaglobin and in 28 cases (23.1%) with GCDFP-15. In the majority of cases, the staining intensity and number of cells staining were higher with mammaglobin than with GCDFP-15. Positive mammaglobin staining was also seen in 44 (8.1%) of 544 nonbreast tumors. Mammaglobin is a more sensitive marker than GCDFP-15 for breast carcinoma; however, it lacks the specificity of GCDFP-15.

The expressions of three mRNA markers were correlated with the results of extensive histopathological examination of a total of 290 axillary lymph nodes from 29 breast carcinoma patients. Included were two established markers for breast cancer (cytokeratin-19 and mammaglobin) and the novel marker DNA methyltransferase 3b (DNMT3b). DNMT3b was significantly overexpressed in breast cancer compared to normal breast tissue. The expression of the three markers in axillary lymph nodes was determined using quantitative real-time RT-PCR. DNMT3b expression showed a specificity of more than 99%, which was comparable to that of cytokeratin-19 and better than that of mammaglobin. The sensitivity of RT-PCR relative to histopathology was highest for cytokeratin-19 (96%), followed by DNMT3b (88%) and mammaglobin (68%). The overall agreement of histological and RT-PCR results was 96-99%. The results indicate that expression analysis of marker genes by quantitative RT-PCR can be a useful tool for lymph node diagnosis in breast cancer.

As many as 30% of node-negative breast cancer patients relapse within five years, suggesting that current histological detection methods are inadequate for identifying metastatic disease. Detecting small number of cancer cells in the breast tissue or lymph node by reverse transcription-polymerase chain reaction (RT-PCR) assays using a combination of tissue and cancer specific markers might be very useful in the early detection or monitoring of the treatment. Mammaglobin is a member of the uteroglobin gene family and appears to be expressed only in breast tissue. Carcinoembryonic antigen has been the preferred molecular marker for detection of micro metastases in lymph nodes in almost all carcinomas.Samples were collected from randomly chosen breast cancer patients undergoing modified mastectomy or breast conserving surgery between September 2003 and July 2004. RT-PCR was applied to study the expression of MMG and CEA markers. Breast cancer micrometastases in axillary lymph nodes were also assessed.The MMG marker was positive in 9/10 normal breast tissues, 3/3 breast fibroadenomas and 37/39 of breast carcinoma tissues, giving an overall sensitivity of 94%. The sensitivity was 80% for metastatic lymph node samples. On the other hand CEA showed 95% sensitivity for malignant breast tumors and 100% sensitivity for metastatic lymph nodes.RT-PCR using a combination of MMG and CEA markers is a powerful tool to complement current routine histopathology techniques for detection of breast cancer metastasis in axillary nodes.

Background In this study we have validated the feasibility of detecting disseminated tumor cells (DTC) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Bone marrow samples from a large cohort of patients with breast cancer were analyzed for the presence of DTC by immunocytochemistry (ICC) or a molecular-based method. Patients and Methods Bone marrow samples were collected from 170 patients with breast cancer with stage I-IV disease before the initiation of any local or systemic treatment. Staining for cytokeratin (CK)—positive cells was performed with the Epimet® kit. Disseminated tumor cells were also quantified by measuring relative gene expression for CK19 and mammaglobin (MAM) using a quantitative RT-PCR detection method. The mean follow-up time was 30 months. Kaplan-Meier analysis was used for predicting overall survival. Results Despite an excellent quantitative correlation and qualitative concordance between ICC and RT-PCR, survival analysis suggested an improved prognostic significance of DTC as detected by quantitative RT-PCR. Univariate survival analysis computed a relative risk of death of 2.87 for women with ICC-positive cells in the bone marrow, as compared with those without positive cells. The relative risk for women with RT-PCR—positive bone marrow was even higher: 3.5 (CK19) and 3.39 (MAM). In multivariate analysis, bone marrow CK19 was a stronger prognostic factor than bone marrow ICC. Conclusion Reversetranscriptase polymerase chain reaction—detected DTC is shown to be prognostically significant in untreated patients with breast cancer. Furthermore, it seems to be a more sensitive method for detecting DTC in bone marrow samples when compared with ICC.

Background Mammaglobin A (SCGB2A2) and lipophilin B (SCGB1D2), two members of the secretoglobin superfamily, are known to be co-expressed in breast cancer, where their proteins form a covalent complex. Based on the relatively high tissue-specific expression pattern, it has been proposed that the mammaglobin A protein and/or its complex with lipophilin B could be used in breast cancer diagnosis and treatment. In view of these clinical implications, the aim of the present study was to analyze the expression of both genes in a large panel of human solid tumors (n = 309), corresponding normal tissues (n = 309) and cell lines (n = 11), in order to evaluate their tissue specific expression and co-expression pattern. Methods For gene and protein expression analyses, northern blot, dot blot hybridization of matched tumor/normal arrays (cancer profiling arrays), quantitative RT-PCR, non-radioisotopic RNA in situ hybridization and immunohistochemistry were used. Results Cancer profiling array data demonstrated that mammaglobin A and lipophilin B expression is not restricted to normal and malignant breast tissue. Both genes were abundantly expressed in tumors of the female genital tract, i.e. endometrial, ovarian and cervical cancer. In these four tissues the expression pattern of mammaglobin A and lipophilin B was highly concordant, with both genes being down-, up- or not regulated in the same tissue samples. In breast tissue, mammaglobin A expression was down-regulated in 49% and up-regulated in 12% of breast tumor specimens compared with matching normal tissues, while lipophilin B was down-regulated in 59% and up-regulated in 3% of cases. In endometrial tissue, expression of mammaglobin A and lipophilin B was clearly up-regulated in tumors (47% and 49% respectively). Both genes exhibited down-regulation in 22% of endometrial tumors. The only exceptions to this concordance of mammaglobin A/lipophilin B expression were normal and malignant tissues of prostate and kidney, where only lipophilin B was abundantly expressed and mammaglobin A was entirely absent. RNA in situ hybridization and immunohistochemistry confirmed expression of mammaglobin A on a cellular level in endometrial and cervical cancer and their corresponding normal tissues. Conclusion Altogether, these data suggest that expression of mammaglobin A and lipophilin B might be controlled in different tissues by the same regulatory transcriptional mechanisms. Diagnostic assays based on mammaglobin A expression and/or the mammaglobin A/lipophilin B complex appear to be less specific for breast cancer, but with a broader spectrum of potential applications, which includes gynecologic malignancies.

Methods for identifying sentinel lymph nodes (SNs) and their clinical significance have been established. Recent advances in molecular immunology have enabled the analysis of precise immune responses. The objective of the current study was to clarify the dendritic cell (DC) maturation, T-helper type 1 (Th-1) and Th-2 responses, and regulatory T-cell responses of SNs in patients with breast carcinoma.SNs and non-SNs were identified by radioguided and blue dye-guided methods in 70 consecutive patients with clinically lymph node negative (N0) breast carcinoma. Lymphocytes were collected from SNs and non-SNs and were subjected to flow cytometric analysis (FCM) using antibodies of CD83-fluorescein isothiocyanate (FITC), CD80-phycoerythrin (PE), CD86-PE, CD40-PE, human leukemic D-related antigen (HLA-DR)-FITC, CD4-FITC, and CD25-PE. Total RNA was extracted from SNs and non-SNs, and the expression of CD83, interleukin 12p40 (IL-12p40), interferon gamma (IFN-gamma), IL-4, IL-10, and Foxp3 was evaluated by using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The immunologic status of SNs was analyzed further with regard to micrometastases, which were identified as negative microscopically but positive according to an RT-PCR analysis that was specific for mammaglobin.SNs were detectable in 70 of 71 consecutive patients (98.6%) with clinically N0 breast carcinoma. Fourteen of 70 patients (20.0%) had positive metastasis in SNs. When SNs were compared with non-SNs in 56 metastasis-negative patients, FCM revealed that HLA-DR-positive, CD80-positive, CD86-positive, and CD40-positive cell populations were decreased significantly in SNs. RT-PCR analysis demonstrated that, among 44 patients with metastasis-negative SNs, the expression levels of CD83 and IFN-gamma mRNA were significantly lower in SNs compared with non-SNs. Immunologic parameters also were compared between 44 metastasis-negative SNs and 14 metastasis-positive SNs. The metastasis-positive SNs demonstrated significantly higher expression of CD83, IL-12p40, IFN-gamma, IL-10, and Foxp3 mRNA than the metastasis-negative SNs. Correction of micrometastasis detected by mammaglobin enhanced these differences consistently.In patients with breast carcinoma, cellular immune responses, from DC maturation to Th-1 responses, appeared to be less active in SNs compared with non-SNs before metastasis developed. Once metastasis was established in SNs, DC maturation was triggered and was followed by the up-regulation of Th-1 responses, which may reflect antigen-specific immune responses in SNs. Unlike DC maturation and Th-1 responses after metastasis in SNs, up-regulation of Th-2 and regulatory T-cell responses developed in parallel.

We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.

BACKGROUND: Mammaglobin is expressed mainly in mammary tissue, overexpressed in breast cancer (BC) and rarely in other tissue. The aim of this study was to assess the sensitivity and specificity of transcript MGB1 detection and to evaluate the role of MGB1 as potential clinical marker for the detection of disseminated cancer cells in the blood of BC patients. PATIENTS AND METHODS: A consecutive series of 23 BC tissues, 36 peripheral blood BC samples and 35 healthy peripheral blood samples was prospectively recruited to investigate MGB1 expression by means of a quantitative Real Time RT-PCR assay. RESULTS: MGB1 overexpression in tissue samples of BC patients is significantly associated only with high level of Ki67 (P

Genes that are expressed in a highly tissue- or disease-specific manner provide possible targets for therapeutics, early detection of cancer, and monitoring of disease burden during and after treatment. Further, genes of this type that code for secreted or shed proteins may allow for serum detection of the product facilitating our ability to specifically detect the cancer in all circumstances. To this end, we are working towards identification and characterization of such genes that are specifically expressed in breast epithelium. In the current study, we have measured the expression of two markers that emerged from a screen of the Incyte LifeSeq Database and were subsequently shown to be highly restricted to breast epithelium termed BU101 (also called Lipophilin B) and BS106 (small mucin-like protein). These two novel markers were compared with two other candidate markers, Mammaglobin and Cytokeratin 19 (CK19). Utilizing quantitative real-time PCR, we compared the expression of these four genes in a series of 95 primary breast cancers, 9 lymph nodes from breast cancer patients, 13 lymph nodes from non-cancer patients and 10 normal breast tissues. Cytokeratin was shown to be highly sensitive in detecting all breast cancers, while BU101, BS106 and Mammaglobin were more restricted. While no one of the these markers efficiently detects all breast cancers, a combination of two or more could achieve a very high sensitivity in assaying for circulating or occult breast cancer cells.

Purpose: Early detection of breast cancer has implications for the management and treatment of patients with this disease. Currently, there exist no highly sensitive and specific serologic biomarkers for detection of breast cancer. Mammaglobin is predicted to be a secreted protein, and expression of this gene seems to be highly specific in breast cancer. The present studies were undertaken to develop the mammaglobin protein as a serum biomarker for detection of breast cancer. Experimental Design: We characterized the mammaglobin protein as a secreted, 14- to 21-kDa species, which is likely post-translationally processed based on its predicted 7-kDa size. Immunostaining for mammaglobin was conducted. An ELISA was developed for the detection of the mammaglobin protein in serum, and levels were compared between women with and without breast cancer. A receiver operating characteristic curve was used to show sensitivity and specificity for cut points on the continuous mammaglobin scale. Results: The protein was detectable by immunostaining in 72% of breast tumors and not in other tumor types. The ELISA was highly sensitive and specific for detection of mammaglobin protein in tissue culture fluids of breast cancer cells and sera of breast cancer patients. The ELISA differentiated healthy women from those with breast cancer with accurate, repeatable results across time and under varying storage conditions. Conclusion: Our results indicate that mammaglobin, as measured by the ELISA, holds significant promise for breast cancer screening with the realistic potential to impact management of this disease.

We designed a low-density microarray carrying 132 DNA capture sequences highly specific for genes known to be differentially expressed among breast tumors and BCC lines or associated with specific tumor properties (cell-cycle alteration, proteolysis, adhesion, hormone sensitivity, etc). We analyzed gene expression in 11 BCC lines among which 6 had already been extensively studied (BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-453, T-47D) and 5 were still poorly characterized (Evsa-T, IBEP-1, IBEP-2, IBEP-3, KPL-1). Some data obtained were verified or extended by real-time polymerase chain reaction (real-time PCR), Northern blotting, Western blotting, immunohistochemistry and cell growth studies. Clustering analysis of the low-density microarray data allowed the sorting of BCC lines into two classes and supported a major discriminatory role for ER alpha, confirming data from previous studies. A few genes that are highly and specifically expressed in one cell line were identified, such as MGB1 (mammaglobin 1) in Evsa-T cells, and PIP (prolactin-inducible protein) in MDA-MB-453 BCC, suggesting an apocrine origin for these latter cells. Two BCC lines (IBEP-1 and IBEP-3) that had been previously characterized as ER alpha-negative, were classified by the low-density microarray among ER alpha-positive lines (MCF-7, T-47D, IBEP-2, BT-474, KPL-1) and were indeed confirmed as receptor-positive (at both mRNA and protein levels) and hormone-responsive cells. In conclusion, our results support the utility of a low-density microarray approach in cases where the cost and exhaustiveness of high-density microarrays may constitute a drawback; for instance, in obtaining a rapid phenotype evaluation in cell populations freshly isolated from breast tumors.

The objective of this study was mainly to develop and evaluate a membrane array-based method simultaneously detecting the expression levels of a multiple mRNA marker panel in the peripheral blood for used in complementary breast cancer diagnosis. The mRNA markers employed included cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA), c-Met, Her2/neu, and mammaglobin (hMAM). The specimens of peripheral blood were collected from 80 healthy women and 102 female patients with breast cancer. The expression levels of molecular markers were evaluated by real-time Q-PCR and membrane array. Data obtained from real-time Q-PCR and membrane array were subjected to linear regression analysis, revealing that there was a high degree of correlation between the results of these two methods (r=0.979, P

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-alpha, IL-1beta) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells.

The breast cancer-associated gene mammaglobin is a member of the secretoglobin protein family and has demonstrated its utility as a breast cancer marker. However, the transcriptional regulation of mammaglobin has not been well-characterized. In this report, we used luciferase reporter assays to identify the 200 bp directly 5' of the transcriptional start site as the minimal promoter region of mammaglobin. Sequence scanning indicated that two PEA3 transcription sites were possibly involved in mammaglobin transcription. By transfecting a PEA3 expression vector into breast cancer cell lines MDA-MB-415 and MCF-7, we determined that exogenous PEA3 was able to drive transcription. Mutational analysis indicated that each PEA3 site was functional. Our reporter system and electrophorectic mobility shift assays (EMSAs) also identified the involvement of a unique repetitive element in mammaglobin transcription. Finally, AP-1 was determined via luciferase assays to be involved in regulating non-PEA3 dependent transcription. Elucidating these cis-acting elements will impact our understanding of transcription of normal breast and breast cancer-associated genes.

To the Editor: In a recent issue of Clinical Cancer Research , two articles ([1][1], [2][2]) addressed the use of PCR to detect micrometastases from breast cancer either in lymph nodes ([1][1]) or in peripheral blood ([2][2]). Both articles found mammaglobin ( mam ) to be an excellent marker for

A breast cancer-associated antigen, mammaglobin-A, is specifically expressed in 80% of primary breast tumors. The definition of immune responses against this highly expressed breast cancer-specific antigen should be of great value in the development of new therapeutic strategies for breast cancer. Thus, the purpose of this study was to identify HLA-A2-restricted mammaglobin-A-derived epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL). We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A2 molecule (Mam-A2.1-7) by means of a HLA class I-peptide binding computer algorithm from the BioinformaticsMolecular Analysis Section of the National Institutes of Health. Subsequently, we determined that CD8+ CTLs from breast cancer patients reacted to the Mam-A2.1 (83-92, LIYDSSLCDL), Mam-A2.2 (2-10, KLLMVLMLA), Mam-A2.3 (4-12, LMVLMLAAL), Mam-A2.4 (66-74, FLNQTDETL), and Mam-A2.7 (32-40, TINPQVSKT) epitopes using an IFN-gamma ELISPOT assay. Interestingly, healthy individuals also showed high reactivity to the Mam-A2.2 epitope. Two CD8+ CTL lines generated in vitro against TAP-deficient T2 cells loaded with the candidate epitopes showed significant cytotoxic activity against the Mam-A2.1-4 epitopes. These CD8+CTL lines recognized a HLA-A2+breast cancer cell line expressing the Mam-A2.1 epitope. In addition, DNA vaccination of HLA-A2+/human CD8+ double-transgenic mice with a DNA construct encoding the Mam-A2.1 epitope and the HLA-A2 molecule induced a significant expansion of epitope-specific CD8+ CTLs that recognize the same HLA- A2+/Mam-A2.1+ breast cancer cell line. In conclusion, these results demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and prevention of breast cancer.

Mammaglobin is a gene that is expressed almost exclusively in the normal breast epithelium and human breast cancer. It is a member of the secretoglobin gene family and forms a heterodimer with lipophilin B. We have focused on the tissue-specificity of mammaglobin as a potential mechanism for the specific killing of breast cancer cells. By elucidating the promoter region of mammaglobin, we hope to utilize this site as a method for turning on the apoptosis inducer gene, Bax, in breast cancer cells. The Bax gene will only be expressed at levels necessary to induce apoptosis in mammaglobin positive cells. This would include > 80% of all breast cancer cells and some normal breast epithelium. This type of targeted killing could be conceptualized as a biochemical mastectomy; that is, genetic ablation of breast tumor cells and perhaps non-malignant breast epithelium while preserving the adipose and stromal components of the breast. Work is also being done to address the binding specificity of the secreted mammaglobin protein. There is early evidence that the mammaglobin heterodimer may in fact bind to breast and breast cancer cells. If this finding is validated, this creates the possibility that mammaglobin can be tagged with a radioisotope or a toxin, so that binding of the tagged-mammaglobin complex results in the specific killing of that breast cancer cell. Finally, mammaglobin is being explored as a target for immune-based interventions. In vitro studies have demonstrated that T cellmediated immune responses can be induced against mammaglobin-derived peptides expressed by MHC molecules on tumor cells and antigen-presenting cells. In summary, mammaglobin displays several unique features that make it a promising target for intervention.

Background: The aim of this study was to examine the potential usefulness of a mammaglobin multigene reverse transcription-PCR (RT-PCR) assay and a mammaglobin sandwich ELISA as diagnostic tools in breast cancer. Methods: We studied peripheral blood samples from 147 untreated Senegalese women with biopsy-confirmed breast cancer and gathered patient information regarding demographic, and clinical staging of disease. The samples were tested for mammaglobin and three breast cancer-associated gene transcripts by a multigene real-time RT-PCR assay and for serum mammaglobin protein by a sandwich ELISA assay. Results: In 77% of the breast cancer blood samples, a positive signal was obtained in the multigene RT-PCR assay detecting mammaglobin and three complementary transcribed genes. Fifty samples from healthy female donors tested negative. Significant correlations were found between mammaglobin protein in serum, presence of mammaglobin mRNA-expressing cells in blood, stage of disease, and tumor size. Circulating mammaglobin protein was detected in 68% of the breast cancer sera, and was increased in 38% in comparison with a mixed control population. The RT-PCR assay and the ELISA for mammaglobin produced a combined sensitivity of 84% and specificity of 97%. Conclusion: The ELISA and RT-PCR for mammaglobin and mammaglobin-producing cells could be valuable tools for diagnosis and prognosis of breast cancer.

Mammaglobin A, in contrast to other factors, is a breast specific member of uteroglobin gene family. Expression is restricted to normal and neoplastic breast epithelium. A highly homologous mammaglobin B is not specific to breast tissue. In this pilot feasibility study we examined expression of both markers for minimal residual disease in the bone marrow of patients with breast cancer. We obtained bone marrow aspirates of 34 patients with stage I (41%), II (56%) and III (3%) breast cancer who underwent either immediate complete resection of the tumor or neoadjuvant therapy with subsequent curative surgery. mRNA was isolated using QIAamp RNA blood mini kit (Qiagen). Subsequently two-step nested RT-PCR for the expression of mammaglobin A and mammaglobin B was performed. Mammaglobin A was detected in samples from 4 (12%) out of 34 patients. None of the specimens was positive for mammaglobin B. With a median follow-up of 21 month we observed only 2 recurrences, one in patient with mammaglobin A positive bone marrow.RT-PCR assay for mammaglobin A may be a useful tool for detection of occult breast cancer cells in the bone marrow. Clinical and prognostic relevance of minimal residual disease should be further investigated.

The nuclear pore complex protein Nup88 is overexpressed in tumor cells. Immunohistochemical studies have shown that this overexpression is linked to higher aggressiveness of colorectal carcinoma and to enhanced metastatic potential of melanoma cells. However, the antibodies so far developed against Nup88 have the drawback of recognizing a number of other, up to now unspecified antigens besides Nup88. For this reason, we devised the present study on Nup88 expression at the mRNA level. RNA was extracted from fresh tumor tissue corresponding to 122 breast cancer patients. Nup88 mRNA expression was measured by means of differential RT-PCR, standardizing against a constitutive internal control gene (beta-actin). The results were dichotomized into "high" and "low" expression levels, using the median value as cut-off. High Nup88 mRNA expression levels correlated significantly with ductal and tubular histology (p = 0.012), histologic and nuclear grade 3 of tumors (p0.001), absence of hormone receptor expression (p0.001), expression of the c-erb-B2 oncogene (p0.001), expression of mutant p53 protein (p0.001), high proliferation (defined by Ki67 labeling index20%, p0.001), DNA aneuploidy (p0.001) as well as the most important ominous clinical prognostic factor, axillary node invasion (p0.001). We also found an inverse correlation (p0.001) with expression of the H-MAM (mammaglobin) gene, a marker of low biologic and clinical aggressiveness of breast cancer. All of these factors, without exception, define a highly aggressive tumor phenotype. These findings appear to be specific to Nup88 and not to nuclear pore proteins in general. Indeed, analysis of Nup107 (which is a limiting component of the nuclear pore complex) under the same conditions in the same tumors did not yield comparable results.

We describe a new one-step RT-PCR assay for the detection of the mammaglobin (MGB1) gene transcript in the peripheral blood of breast cancer patients. With this approach, the MGB1 transcript could be detected in the peripheral blood of 22 of 54 (41%) breast cancer patients prior to any therapy. This method, using specific primers for cDNA synthesis, proved to be more sensitive (10−6 to 10−11, usually 10−7) than previously reported methodologies. This increased sensitivity was achieved without compromising specificity, as the MGB1 transcript was not detected in 38 blood samples of healthy donors and in only 1 of 18 blood samples of patients presenting with hematologic malignancies. A positive correlation was seen between MGB1 positivity and breast cancer stage: 0/3 (0%) in stage 0, 3/13 (23%) in stage I, 6/17 (35%) in stage II, 5/10 (50%) in stage III, 8/11 (73%) in stage IV (p = 0.003). The prognostic and therapeutic implications of MGB1 positivity by one-step RT-PCR in the peripheral blood of breast cancer patients, especially in clinically localized disease (stages I and II), should be evaluated after long-term clinical follow-up of these patients. © 2003 Wiley-Liss, Inc.

Background Nasal polyps are a common problem that is difficult to diagnose and treat, in part because the cause of nasal polyposis is unknown. Although information on the pathogenesis of polyposis is lacking, there are reports suggesting that a genetic predisposition underlies this disorder. Objective We sought to better understand the basis of nasal polyposis associated with allergic rhinitis. We hypothesize that the expression of unique genes is associated with the nasal polyposis phenotype. Methods We examined 12,000 human genes transcribed in the nasal mucosa of patients with allergic rhinitis with and without nasal polyps. Biopsy specimens of the mucosa of patients with and without polyps were obtained after the patients refrained from the use of topical or systemic steroid therapy for 2 weeks. Results Thirty-four genes were differentially expressed between the patient groups, including those for inflammatory molecules and putative growth factors. The greatest differential expression identified by the array analysis was for a group of genes associated with neoplasia, including mammaglobin, a gene transcribed 12-fold higher in patients with polyps compared with control patients with rhinitis alone. Quantitative RT-PCR confirmed this differential expression and documented that the number of mammaglobin mRNA copies is actually 64-fold greater in tissues of patients with polyps versus control patients. The specificity of mammaglobin protein expression was evaluated by means of immunohistochemistry, which showed specific staining in nasal polyp mucosal goblet cells only in patients with polyps. Conclusion These data suggest that nasal polyposis involves deregulated cell growth, using gene activation in some ways similar to a neoplasm. In addition, mammaglobin, a gene of unknown function associated with breast neoplasia, might be related to polyp growth.

Mammaglobin, known for its mammary tissue specificity, has been discussed as a promising diagnostic marker in breast cancer for almost 10 years. In particular, the application of mammaglobin RT-PCR to detect disseminated breast cancer cells has been reported. More than 25 publications evaluate the detection of mammaglobin mRNA in lymph node, blood, and bone marrow specimens of breast cancer patients. Recently, structural details about the mammaglobin complex have been discovered, and these findings can be implemented to optimize detection of the secreted protein. This review summarizes the findings of almost 50 published studies and the current knowledge about the diagnostic utility of mammaglobin.

Mammaglobin (SCGB2A2) is a breast-specific member of the secretoglobin (SCGB) gene family. SCGB2A2 has previously been found overexpressed in breast tumors but possible associations between its expression and established prognostic tumor characteristics such as the levels of estrogen and progesterone receptors have not yet been investigated. We evaluated SCGB2A2 expression at the mRNA and at the protein level by reverse-transcription polymerase chain reaction and immunocytochemistry in 52 and 32 breast tumors, respectively. Both SCGB2A2 mRNA and protein expression were significantly higher in estrogen-receptor-positive compared to estrogen-receptor-negative tumors (Mann- Whitney rank sum test, p = 0.04; chi-square test, p = 0.01; respectively). In contrast, SCGB2A2 expression did not correlate with progesterone receptor levels or Nottingham grade. As estrogen and antiestrogen treatment of estrogen-positive breast cancer cell lines does not modify SCGB2A2 expression we suggest that SCGB2A2 may be a new independent breast cancer prognostic marker.

Human mammaglobin (hMAM) mRNA is considered to be a promising candidate for a sensitive molecular marker for breast cancer. In this study, we attempted to relate the presence of hMAM mRNA in the peripheral blood with certain established clinico-pathological features of breast cancer in order to validate its clinical utility. A total of 139 subjects including 79 with localized cancer, 33 with metastatic disease, and a control group of 27 individuals were studied. hMAM mRNA expression was assessed by reverse transcriptase polymerase chain reaction on cells from peripheral blood. The expression of hMAM mRNA was found in 0 of the 27 control subjects, 1 of the 8 stage 0 (12.5%) patients, 4 of the 16 stage I (25%) patients, 13 of the 40 stage II (32.5%) patients, 5 of the 15 stage III (33.3%) patients, and 18 of the 33 (54%) cases of metastatic disease. There was a statistically significant (P=0.045) difference in frequency between patients with localized disease (29%) and those with metastatic disease. Although trends of increasing frequency of hMAM mRNA expression existed in patients with unfavorable prognostic factors, including primary tumor size, T stage, N stage, overall stage, presence of angiolymphatic permeation, negative estrogen receptor, high S-phase fraction (>7%), and aneuploid DNA index, none of the differences was significant. In conclusion, the clinical utility of hMAM mRNA may not be in screening or staging of breast cancer, but in following patients after surgery to detect recurrence. Further evaluation of hMAM mRNA in combination with other molecular markers to follow post-operative breast cancer patient is warranted. (Cancer Sci 2003; 94: 99–102)

We conducted a study to compare the expression of human mammaglobin (hMAM) mRNA in breast cancer patients’ peripheral blood with serum carcinoembryonic antigen (CEA) and CA 15.3. A total of 33 metastatic breast cancer patients were enrolled. The blood samples were used to test the expression of hMAM mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and CEA, CA 15.3 by radioimmunoassay. The serum CEA and CA 15.3 levels were elevated in 17 (51%) and 23 (69%) of the patients, respectively. When combined CEA with CA 15.3, the sensitivity rate raised to 78%. hMAM mRNA was detected in 18 (54%) of the 33 patients. When combined hMAM mRNA with CEA or CA 15.3, the sensitivity rate were 81% and 90%, respectively (P=0.045). In conclusion, the hMAM mRNA RT-PCR can be an adjunct in detecting metastatic breast cancer.